Novel gp41 antigens

ABSTRACT

The present invention deals with a modified polypeptide comprising three contiguous segments N, L and C represented by the formula N−L−C and comprising: a N-helix region of gp41 (N), a C-helix region of gp41 (C), and a connecting loop comprising a synthetic linker (L) between the N and C-helices, the linker replacing amino acids 593-617 of gp41, the numbering scheme being based upon the prototypic isolate HIV-1 HxB2 Clade B strain, said polypeptide comprising the calveolin-1 neutral izing and 98.6 D epitopes, but not 2F5 and 4E10 epitopes, not the fusion peptide, the polypeptide having a minimal immunogenic cross-reactivity with human interleukin 2 (IL2).

BACKGROUND OF THE INVENTION

The instant invention is directed to a soluble and stabilized form inaqueous media of the envelope glycoprotein gp41 of HIV-1 suitable forinducing an immune response against a human immunodeficiency virus type1 (HIV-1), pharmaceutical compositions comprising said gp41, a method oftreatment against a human immunodeficiency virus, and/or HIV relateddiseases or disorders.

HIV-1 encodes a 160 kDa envelope glycoprotein (gp160) precursor, whichis proteolytically cleaved into the exterior (gp120) and transmembrane(gp41) glycoproteins.

In the glycoprotein mature envelope, the gp120 glycoprotein remainsassociated with the gp41 ectodomain through a noncovalent interaction.The native HIV-1 envelope glycoproteins exist predominantly as trimersat the surface of the viral membrane, which consists of three gp120 andthree gp41 subunits and are anchored in the viral or infected cellmembrane by the gp41 transmembrane region.

It has been shown that the binding of gp120 to the CD4 receptor inducesconformational changes that promote subsequent interaction with one of anumber of chemokine receptors (CXCR4, CCR5 . . . ). These binding eventstrigger conformational changes in gp41. In particular, studies by X-raycrystallography and nuclear magnetic resonance indicate that the viralenvelope glycoprotein gp41 exists in at least three conformations, anative conformation (spike), a prefusogenic metastable conformationwhich is converted to a thermostable fusogenic “three hairpin”conformation following a triggering event, such as binding of HIV-1virus particle to the membrane of target cells.

So, the binding of gp120 to cellular coreceptors induces the gp41conversion from a prefusogenic form to a fusogenic form.

The linear organization of the gp41 includes a fusion peptide, anectodomain (a N-terminal coiled-coil, a disulfide-bonded loop region,and a C-terminal a-helical segment) and a transmembrane domain.

In the fusogenic six-helix bundle of the gp41, three N-terminal helicesform a trimeric coiled-coil, and three C-terminal helices pack in thereverse direction into three hydrophobic grooves on the surface of thecoiled-coil. This helical-hairpin structure corresponds to thefusion-active conformation of gp41. Because the transmembrane anchor andthe fusion peptide of the gp41 ectodomain are embedded in the viral andtarget cell membranes, respectively, the formation of the fusogenichairpin structure results in the colocalization of the two membranes andthus overcomes the energy barrier for membrane fusion.

The envelope glycoproteins of HIV-1 represent the only realistic viraltarget for vaccine-induced neutralizing antibody responses because theypromote viral membrane fusion through receptor-mediated conformationalchange and they are expressed on the surface of both virions andinfected cells. Monomeric HIV-1 gp120 and derivatives were initiallyconsidered to be principal vaccine candidates. However, HIV-1 gp120 ishighly variable and has repeatedly proven to be an immunogen ineffectiveat eliciting neutralizing antibodies against clinical HIV-1 isolates.Few of the antibodies raised by gp120 monomers effectively bindassembled HIV-1 envelope glycoprotein trimers.

In contrast, gp41 is an extremely immunogenic glycoprotein, inducingantibodies in essentially all HIV-infected individuals.

The ectodomain of gp41 is the most conserved region of the HIV-1envelope, membrane protein which otherwise exhibits considerable geneticdiversity even among closely related isolates.

Furthermore, the gp41 performs a critical role in maintaining theconformation and infectivity of the HIV-1 virions.

The antibodies targeting the six-helix bundle (fusogenic form) andprehairpin (prefusogenic form) structures arrest fusion under certainconditions. Antibodies having access to prehairpin and six-helix bundlesconformations of gp41 would be capable of inhibiting gp41-mediatedfusion. Furthermore, the six-helix bundle is an extremely stablestructure.

Those observations allow considering the gp41 six-helix, under amodified form or not, as an attractive target for drugs and vaccinedevelopment.

In U.S. Pat. No. 6,455,265, it was shown that some gp41 derivativescould be particularly efficient for obtaining vaccines for preventingthe pathogenic effects related to a HIV retroviral infection, with theproviso that the corresponding polypeptides have epitopes having amodified antigenicity so as to obtain a differential immune responsewith respect to the viral envelope and some self-proteins.

More precisely, it was discovered that conserved and immunodominantregions of the retroviral envelope could be responsible for harmfulautoimmune phenomena, particularly in the case of the gp41 retroviralenvelope. It was observed that certain immunodominant regions of thegp41 exhibit three-dimensional structural analogies and/orcross-reactivities with certain regions of some proteins of the humanimmune system, and in particular the interleukin 2 (IL-2).

Accordingly, it was proposed in U.S. Pat. No. 6,455,265 modifiedpolypeptides obtained by modifying the antigenicity of the concernedepitope of the envelope protein, in order to obtain a differentialimmune response with respect to the viral envelope protein and theseproteins of the human immune system, in particular IL-2.

According to WO2005/01033, such modified polypeptides with at least oneantigenic region of native gp41 protein of HIV-1 have been disclosed

Generally, synthetic gp41 can be produced in transfected baculovirus ormammalian cells but the yield is lower than in E. coli. Furthermore, theglycosylation in baculovirus or mammalian cells is different from theglycosylation of human cells and is not necessary for the immunogenicityof the protein. Gp41 is in fact very immunogenic without glycosylation.

However, full length or shorter recombinant HIV-1 ectodomain of gp41produced in E. coli generally forms insoluble precipitates (aggregatesof gp41 trimeric form) in aqueous media at neutral pH.

There is still a need to produce high levels of gp41 proteins that maybe devoid of immunodominant region that trigger antibodies with noneutralizing activities but keeping important gp41 regions to focus theimmune response on relevant epitopes that retain their overallimmunogenic activity.

However there is still a need for a vaccine that allows for inducing aversatile immune response against HIV infection, and in particularHIV-type 1 infection. There is also a need for the development ofnon-clade B vaccines, such as, for example, clade C strains.

There is also a need for the development of a vaccine with broadinhibitory spectrum allowing for cross-clade inhibition.

There is a need for a vaccine allowing to induce an innate and/or ahumoral and/or cellular immune response against HIV-1 infection.

There is a need for a vaccine allowing to induce an immune responseagainst HIV infection at the mucosal surface level and/or at the bloodlevel.

There is a need for a vaccine suitable for inducing mucosal IgA and/orantibodies and/or systemic IgA and/or IgG antibodies capable ofinterfering with HIV entry across the mucosa and early cell infectionunder the mucosa.

There is a need for a vaccine suitable for inhibiting or reducing HIVentry across mucosal tissues, e.g. vaginal mucosal tissues throughvarious mechanisms such as transcytosis and ADCC (Antibody Depedent CellCytotoxicity).

It is an object of the invention to satisfy to all those above-mentionedneeds.

SUMMARY OF THE INVENTION

The instant invention is more precisely directed to propose stabilizedhydrosoluble forms of gp41 protein.

Unexpectedly, the inventors have discovered that it was possible todecrease significantly any immunodominant cross reaction with someproteins of the human immune system, the hydrophobicity of the loop, asto increase the solubility and the stability of the gp41 derivatives,resulting in a trimeric soluble form of gp41, without altering itsimmunogenic reactivity. In addition, according to a preferred, but nonexclusive embodiment said polypeptides are easily purified and attachedto a vehicule suitable for inducing an immune response against a humanimmunodeficiency virus, for instance a virosome.

DETAILED DESCRIPTION OF THE INVENTION

One primary object of the present invention is to design other modifiedpolypeptides having an improved stability, in monomeric or oligomericform, while keeping their solubility in aqueous media, in particularonce they are externally attached or linked to a same virosomeparticule.

Another object of the present invention is to design other modifiedpeptides, which once conjugated with a virosome-like particle, mimickthe orientation / presentation of the gp41 protein on native HIV viralmembrane and/or on any HIV infected cell membrane.

Another object of the present invention is to design other modifiedpeptides having effective antigenic possibly immunogenic properties,which makes them possible candidates for prophylaxis treatment againstHIV. Correspondingly, one object of the present invention is anyantigenic and/or immunogenic compound or composition comprising theseother modified peptides.

Another object of the present invention is to design other modifiedpolypeptides effectively eliciting systemic IgG (blood) and possiblycomplementary mucosal IgA toward relevant conserved regions of gp41protein, in particular against cross-clade variants of HIV, for instanceagainst clade B and clade C of HIV1, among which various subtypesthereof.

Another object of the present invention is to design other modifiedpeptides effectively eliciting protective antibodies and generatinglittle if none, non neutralizing antibodies against HIV, or havingbetter or optimally focused antibody response against the conservedregions of gp41.

Another object of the present invention is to design other modifiedpeptides capable of blocking virus translocation across the mucosalbarrier and/or of inhibiting cell infection, thus preventing HIV-1infection.

Another object of the present present invention is to provide for gp41protein like polypeptides capable of being lipidated, i.e. combineddirectly or indirectly at their C-terminal end with a suitable lipid,with a yield compatible for industrialization/production of any virosomeconjugate of same peptide. Another object of the present invention is toprovide for gp41 protein like polypeptides capable of being linked, i.e.externally attached, to virosome-like particles, with a yield compatiblefor industrialization/ production of any conjugate of some peptide.

Within one aspect of the invention there is provided a modifiedpolypeptide comprising three contiguous segments N, L and C representedby the formula N−L−C and comprising: a N-helix region of gp41(N), aC-helix region of gp41(C), and a connecting loop comprising a syntheticlinker (L) between the N and C-helices, the linker replacing amino acids593-617 of gp41, the numbering scheme being based upon the prototypicisolate HIV-1 HxB2 clade B strain, said polypeptide comprising thecalveolin-1 neutralizing and 98.6 D epitopes, no 2F5 and 4E10 epitopes,no fusion peptide and has a minimal interleukin 2 (IL-2) immunogeniccross-reactivity.

A polypeptide according to the invention is hereinafter indifferentlynamed “gp41 derived antigen” or “gp41 according to the invention” or“rgp41”.

The polypeptide according to the present invention almost maintain anative conformation of an interaction between the N- and C-helices andhave the hydrophobicity that provides a soluble and stable trimeric formto said modified polypeptide without substantially altering itsimmunogenic reactivity.

In the meaning of the present invention, the 2F5 epitope corresponds toa specific region of gp41 recognized by the human 2F5 antibody which hasa broad neutralizing activity for diverse primary HIV-1 isolates (TrkolaA. et al., 1995, J. Virol., 69, pp 6609-6617, see FIG. 1).Thismonoclonal antibody recognizes a core epitope of six amino acids withina relatively conserved 16-amino-acid linear sequence (NEQELLELDKWASLWN,SEQ ID No.7) in the ectodomain of gp41 near the transmembrane region ofthe molecule (Parker et al., 2001, J. Virol., 75, pp 10906-10911).

The 4E10 human monoclonal antibody is specific for the transmembraneproximal region of gp41 in a location immediately nearby carboxyterminal to the 2F5 epitope and also has a broad neutralizing activity(Zwick et al., 2001, J. Virol., 75, pp 10892-10905, see FIG. 1).

The 98.6D epitope is located in cluster II region of gp 41 and isrecognized by the 98.6D human monoclonal antibody as described in GornyM. K. et al., 1989, Proc. Natl. Acad. Sci., 86, pp 1624-1628 and XuJ.-Y. et al., 1991, J. Virol., 65, pp 4832-4838.

The calveolin-1 binding domain corresponds to the CBD1 peptide(SLEQIWNNMTWMQWDK, SEQ ID No. 8) in gp-41 (Benferhat et al., 2009, Mol.Immunol. 46(4), pp 705-712). The fusion peptide corresponds to theamino-terminal region of gp41, which is exposed after formation of thecoiled-coil form. This region is inserted into the membrane of thetarget cell, resulting in the fusion of virus and cell membranes; itcorresponds to the region 512-539 of extracellular portion of gp 41(Quintana et al., 2005, JCI; see FIG. 1).

According to the present invention, a polypeptide allows the formationof gp41-trimers and has retained the native gp41 antigenicity andpresents a minimal IL-2 cross reactivity. Such cross reactivity can bedetermined by methods well known to the skilled man in the art such asgp41-ELISA and gp41-dot blot. An example of such a determination ispresented below (see example 3, FIG. 2).

According to the present invention, the expression “retains the nativegp41 antigenicity” or “without altering its immunogenic activity” meansthat a polypeptide according to the invention has almost the same levelof antigenic and/or immunogenic activity as the wild type gp41.

The N and C segments which constitute a polypeptide according to thepresent invention may be derived from any gp41 protein of HIV, includingthe HIV-1 and HIV2 strains, including laboratory strains and primaryisolates. Preferably, these segments are derived from an HIV-1 strain,and in particular from an HIV1 HxB2 strain such as described in SEQ IDNo. 1.

The nucleotide and peptide sequences of a large number of gp41 proteinsare known and available, for example, on the Internet on the sitehttp://www.hiv.lanl.gov/ and also in the corresponding Los Alamoscompendia (HIV Sequence Compendium 2005 Leitner T, Foley B, Hahn B, MarxP, McCutchan F, Mellors J, Wolinsky S, and Korber B, Eds., published byTheoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, NM, LA-UR 06-0680).

Any sequence, as defined above and/or in the claims, into which one ormore conservative mutations (which do not substantially modifyimmunogenicity) have been introduced is also covered by the abovedefinition.

The amino acids are numbered with reference to the sequence of the gp41protein described in FIG. 1 (which amino acid sequence is represented bySEQ ID No.1).

In a more preferred embodiment the polypeptide of the invention is asequence described by SEQ ID No. 17 or by SEQ ID No. 18.

In a further aspect of the invention, the polypeptide also comprises atleast one spacer peptide segment S. In a specific aspect, thepolypeptide of the invention is represented by SEQ ID No. 19 or SEQ IDNo. 20, and respectively named Mo or M1.

Said spacer sequence being useful to obtain a better conjugation, e.g.linking of the polypeptide with a carrier, e.g. a virosome, renderingthe reactive amino acids on which said grafting is done more accessible.

In particular it may allow to move further apart the amino-acid(s) onwhich said grafting is done from the membrane of the virosome.

The composition of said spacer segment, e.g. amino acid sequence canalso be designed in order to help in the production process of apolypeptide according to the invention. In a particular embodiment ofthe invention, said spacer segment can comprise histidine residues thatcan participate to the purification step of the whole polypeptide (seebelow in example 1).

Said spacer peptide comprises at least the amino acid sequence describedby SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.12 at theC-terminal part of the polypeptide of the invention.

Said spacer sequence may also participate in the immunogenicity of apolypeptide according to the invention.

In preferred embodiments the N segment is represented by the amino acids540-592 of gp41, the numbering scheme being based upon the prototypicisolate HIV-1 HxB2 and/or the C segment is represented the amino acids618-664 of gp41 the numbering scheme being based upon the prototypicisolate HIV-1 HxB2.

According to a preferred embodiment, the N segment is the sequencedescribed by SEQ ID No.13 or SEQ ID No. 14 and /or the C segment is thesequence described by SEQ ID No.15.

In a still further aspect of the invention, said L fragment is asequence described by SEQ ID No. 16.

The polypeptides of the invention are able to form trimers.

In another aspect, the present invention deals with an aqueouscomposition comprising a polypeptide of the invention, said polypeptideforming a stable trimers in an aqueous medium

The present invention, in particular as defined in the following claims,encompasses polypeptides equivalent to those previously defined ordescribed, in particular analogues thereof as defined hereunder withreference to other additional antigens suitable for carrying out thepresent invention.

Within the meaning of the invention, the expression “analogue thereofwith respect to a gp41-derived antigen intends to refer to a peptidehaving substantial (at least 85%, in particular at least 90% and moreparticularly at least 95%) amino acid sequence identity or homology(i.e. aminoacid residue replaced by an aminoacid residue of the samefamily, of similar polarity or charge, for example) with the amino-acidsequence of said gp41-derived antigen, and which has similar orconserved biological properties, in particular with respect to thebinding antigen portion of immunoglobulin directed against the gp41protein.

According to the characteristics described above, a polypeptideaccording to the present invention forms soluble trimers in solution.

As such, in a further aspect, the invention deals with an aqueouscomposition comprising a polypeptide according to the invention, saidpolypeptide, forming a trimer in an aqueous medium. In said aqueouscomposition said trimer is stable.

The oligomeric, e.g. trimeric, state of a peptide according to theinvention can be determined by methods well known to those skilled inthe art such a gel filtration for instance FPLC with a separationbetween 3000 and 600 000 Daltons.

The stability of the trimers formed by the peptide of the invention canbe measured by techniques well known to those skilled in the art such asseveral cycles of freeze and thawing of the aqueous compositioncomprising the polypeptide of the invention.

The polypeptides according to the invention are obtained by anyconventional or standard technique of chemical synthesis or of geneticengineering well known by the person skilled in the art.

According to one option, the polypeptides are produced by chemicalsynthesis: they may be synthesized in the form of a single sequence, orin the form of several sub-sequences which are then linked to oneanother. The chemical synthesis may be carried out in solid phase or insolution, these two synthesis techniques being well known to thoseskilled in the art. These techniques are in particular described byAtherton and Shepard in “Solid phase peptide synthesis” (IRL pressOxford, 1989) and by Houbenweyl in “Methoden der organischen Chemie”[Methods in Organic Chemistry] published by E. Wunsch Vol. 15-1 and 11,Stuttgart, 1974, and also in the following articles, which are entirelyincorporated herein by way of reference: P. E. Dawson et al. (Science1994; 266(5186), pp 776-779); G G Kochendoerfer et al. (1999; 3(6), pp665-671); P E Dawson et al. (2000, 69, Annu. Rev. Biochem., pp 923-960).

According to another option, the polypeptides according to the inventionare produced using genetic engineering techniques well known to thoseskilled in the art. When the said polypeptides according to theinvention are produced by genetic engineering, they may comprise, at theNH2-terminal end, an additional methionine residue corresponding to thetranslation of the first initiation codon.

These techniques are described in detail in Molecular Cloning: amolecular manual, by Maniatis et al., Cold Spring Harbor, 1989.Conventionally, the PCR technique is used to produce the DNA sequenceencoding the polypeptides according to the invention in a form which canbe inserted into an expression vector. The expression vector containingthe sequence of interest is then used to transform a host cell whichallows for expression of the sequence of interest. The polypeptidesproduced are then isolated from the culture medium using conventionalchromatography techniques well known to those skilled in the art. Highperformance liquid chromatography (HPLC) is preferably used in thepurification stage. Typically, the cells are collected by centrifugationat the end of culture, and are taken up in a neutral buffer, in order tobe disrupted by any suitable means. The cell lysate is then centrifugedin order to separate the soluble material from the insoluble material.SDS-PAGE analysis of the supernatant and of the pellet fromcentrifugation reveals whether the polypeptide is soluble or not. If thepeptide is insoluble, solubilization is obtained using a buffercontaining urea, guanidine or any other solubilizing agent.Centrifugation at this step makes it possible to remove debris and otherinsoluble products which would hamper the chromatography. The followingstep consists in loading the solubilized molecule onto an affinitycolumn, which may be of the metal chelate type if a plurality ofhistidine residues such as in the linker segment L which can beintegrated onto the polypeptide of interest. The system which enablesthe affinity purification may be varied in nature, such asimmunoaffinity, affinity on cibachron blue, etc. At this stage, thepolypeptide exhibits a degree of purity close to or greater than 80%, inparticular of at least 90%, as may be determined by colorimetry of aSDS-PAGE electrophoresis followed by Coomassie blue staining.Densitometric measurement of the bands makes it possible to quantify thedegree of purity. The degree of purity may also be measured byreverse-phase HPLC, by measuring the area of the various peaks. Anadditional chromatography step may be added in order to further purifythe polypeptide; by way of example, mention may be made of gelfiltration and reverse-phase chromatography.

In a further embodiment, the present invention also concerns apolynucleotide encoding the above defined polypeptides.

The polynucleotides of the present invention include bothsingle-stranded and double-stranded DNA/RNA molecules.

In a specific aspect the present invention, a polynucleotide encoding argp41 according to the present invention is described by SEQ ID No.21 orSEQ ID No. 28.

Additional DNA sequences encoding modified polypeptides, remainingwithin the scope of the present invention, can be readily generated bythose of ordinary skill in the art, based on the genetic code and thepolypeptide sequences described in the present specification.Counterpart RNA sequences can be generated by substitution of U for T.Those skilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, sequence variation is possible amongpolynucleotide molecules coding for the polypeptides according to thepresent invention, in particular the polynucleotide sequences describedin the present specification.

Conversely, any person skilled in the art will recognize that sequencevariation is possible among polypeptides molecules encoded by thepolynucleotides molecules according to the present invention, inparticular the polynucleotide sequences described in the presentspecification, still in view of the degeneracy of the genetic code.

All these variations are encompassed by the invention definition(s) andappended claims, in so far that those variations do not substantiallyalter the structure/conformation, and/or function(s) and/or propertiesof the resulting polypeptide with reference to the ones specificallypreviously and/or hereinafter described.

According to one embodiment of the invention, a polynucleotide sequenceaccording to the invention is directly chemically synthesized (Young Land Dong Q., 2004,-Nucleic Acids Res., April 15; 32(7), Hoover, D. M.and Lubkowski, J. 2002,. Nucleic Acids Res., 30, Villalobos A, et al.,2006. BMC Bioinformatics, June 6; 7:285).

The polynucleotide sequences of the invention thus obtained can beintroduced in a known manner into any appropriate vector which makes itpossible to express said polypeptide, optionally in modified form, inconvenient cell systems.

The polynucleotide sequences thus obtained can be introduced into a hostcell, so as to transform the host and promote expression (e.g.transcription and translation) of the introduced sequence. Vectorsinclude plasmids, phages, etc. Use is preferably made of vectors inwhich the DNA sequence encoding a polypeptide according to the inventionis under the control of a strong promoter, which may or may not beinducible. As an example of a promoter which may be used, mention ismade of the T7 RNA polymerase promoter. The expression vectors mayinclude a selectable marker, such as the ampicillin, tetracycline orother antibiotic resistance genes appropriate for use in humans.Alternatively the transformed cells can be selected thanks to anauxotrophic marker, or any kind of antibiotic-free selection means(complementation of an essential gene previously knocked-out into thehost's genome).

Examples of expression vectors which may be used include the plasmidspET21b, pET30 (Novagen), yeast, bacteria, viral vectors, such as:baculoviruses, and poxviruses.

In order to promote the expression and purification of a polypeptide,according to the present invention, the latter may be expressed in amodified form, such as a fusion protein, and may include not onlysecretion signals, but also additional heterologous functional regions.For example, a region of additional amino acids, particularly chargedamino acids, may be added at the N-terminal of the polypeptide in orderto improve stability and persistence in the host cell.

An object of the invention also deals with an expression vectorcomprising a polynucleotide as described above.

Said vector can be used to transform a host organism, said host organismforming another object of the present invention.

The invention also provides a host cell transformed with said vector.Any host cell conventionally used in combination with the expressionvectors described above may be used, for instance E. coli, 21 (DE3),BLR(DE3), origami 2(DE3), Bacillus or other gram positive hosts such asLactococcus lactis, yeast, baculovirus and eukaryotic cells such as CHOor Vero. Preferred cell expression systems include E. coli such as BL21(DE3).

In another of its aspect, the present invention deals with a conjugate,such conjugate comprises a polypeptide according to the presentinvention.

An in a specific aspect, the polypeptide the invention is conjugatedwith a virosome-like vesicle.

Virosome-Like Vesicle

A virosome-like vesicle suitable for the instant invention comprises atleast virosomal lipids and preferably exhibits fusion membraneproperties.

According to an embodiment, a virosome-like vesicle of the invention maycomprise a unilamellar lipid bilayer.

According to an embodiment, a virosome-like vesicle of the invention maybe a bi- or a multilamellar vesicle.

According to an embodiment, a virosome-like vesicle may have a diametergenerally in the range of 50 to 600 nm, and in particular a diameterfrom 100 nm to 300 nm, and in particular from 200 nm to 400 nm.

Virosome-like vesicles of the invention may be spherical unilamellarvesicles with a mean diameter with approximately 150 nm. Virosome-likevesicles comprise, incorporated into the lipid bilayer, viral membraneproteins with or without fusion properties or fragments thereof.

The expression “fusion proteins or fragments thereof” is intended torefer to proteins or fragments thereof capable of inducing and/orpromoting a fusion reaction between a virosome-like vesicle membrane anda biological membrane of the target cell.

For example, fusion proteins may be influenza membrane glycoproteinssuch as hemagglutinin (HA).

According to an embodiment, at least two different fusion proteins orfragments thereof may be used, that may display distinct fusioncharacteristic. According to another embodiment, distinct fusioncharacteristics may be, for example, different sensitivity totemperature, to ion concentration, to acidity, to cell type and totissue type specificity.

According to an embodiment, a virosome-like vesicle may contain fusionproteins that mediate fusion at two distinct temperatures. According toanother embodiment, hemagglutinin (HA) from different virus strains maybe used to construct a virosome-like vesicle. As an example, HAmolecules from both X-31 and PR8/34 virions may be capable of catalyzingtwo distinct fusion reactions at distinct temperatures.

Fusion proteins with different fusion characteristics may be derivedfrom different influenza strains, or fusion proteins may be derived fromother viruses, such as the vesicular stomatitis virus (VSV) E1 protein,the Semliki Forest virus (SFV) envelope protein complex, or the Sendaivirus F protein.

An antigen coupled to the membrane of a virosome-like vesicle may bedegraded within the endosome and may be presented to the immune systemby MHC class II receptors. An antigen contained within the lumen of avirosome can be delivered to the cytosol of an antigen-presenting cellby membrane fusion and degraded in the cytosol, after which it may bepresented MHC Class I antigens. Cross-presentation of antigens deliveredby virosomes may also occur.

Therefore, a virosome-like vesicle may be able to induce a humoraland/or a cellular immune response.

In particular, a virosome-like vesicle might induce the production ofIgA antibodies, such as secretory IgA, as well as IgG or IgM. Protocolsof preparation are well-known by the skilled person in the art. Suitableprotocols for the preparation of virosomes are described, for example,in WO 2004/045582 or EP 0 538 437, EP 1 633 395, EP 1594466, which areincorporated herein by reference.

According to an embodiment, a virosome-like vesicle according to theinvention may be obtained either from a virosome vesicle as such, orfrom a vesicle resulting from the fusion of a virosome vesicle with aliposome vesicle.

Preparation of virosome vesicles may be made by any known method of theskilled person in the art such as described by Stegmann et al., EMBO J.6, 1987, no. 9, 2651-9, or de Jonge et al., Biochim. Biophys. Acta,1758, 2006, 527-539, incorporated herein by reference. Virosomevesicles, for example, may be reconstituted from original viral membranelipids and viral membrane glycoproteins after solubilization of, forexample, intact influenza virus with octaethyleneglycol mono-N-dodecylether (OEG), sedimentation of the nucleocapsid (the viral glycoproteinsand lipids will remain in the supernatant), and removal of the detergentfrom the supernatant with a hydrophobic resin (Bio-Beads SM2) (StegmannT, et al., EMBO J. 6, 1987 2651-9).

Virosomes may also be reconstituted from original viral membranes bysolubilizing viral membranes with a short-chain phospholipid,sedimentation of the nucleocapsid (only the viral membrane glycoproteinsand lipids will remain in the supernatant), and removal of theshort-chain lipid in the supernatant by dialysis.

After solubilization of the virus with a detergent or short-chainphospholipid, and the removal of the nucleocapsid as described above,antigens or adjuvants, solubilized in detergent or short-chainphospholipid may be added to the supernatant prior to the removal of thedetergent or short-chain lipid, leading to incorporation of the antigenor adjuvant in the virosome so formed. Likewise, lipids solubilized indetergent or short-chain phospholipid, may be added to the supernatantfor inclusion in the virosomal membrane. Preparation of virosomevesicles containing fusion proteins from different viruses may beperformed by mixing supernatants containing solubilized viral membranesas described above, or by adding purified fusion proteins to suchsupernatant, before said removal of detergent or short-chain lipid.

According to one embodiment, a virosome-like vesicle according to theinvention may be obtained from a fusion of a virosome vesicle with aliposome vesicle.

Therefore, according to one embodiment, a virosome-like vesicle of theinvention may comprise virosomal and liposomal lipids. According to oneembodiment, a virosome-like vesicle of the invention may comprise alipid bilayer comprising lipids chosen from cationic lipids, syntheticlipids, glycolipids, phospholipids, glycerophospholipids,glycosphingolipids like galactosylceramid, sphingolipids, cholesteroland derivatives thereof.

Phospholipids may comprise in particular phosphatidylcholine,sphingomyelin, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatide acid, cardiolipin andphosphatidylinositol with varying fatty acyl compositions.

Cationic lipids may be chosen from DOTMA(N-[l-(2,3-dioleylaxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC(N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB(didodecyldimethylammonium bromide) and stearylamine or other aliphaticamines and the like.

The lipids used in the invention may be formulated as small unilamellarliposomes in a mixture with DOPE (dioleoylphosphatidyl ethanolamine)that is widely used as helper lipid to facilitate disruption of theendosomal membrane.

According to another embodiment, co-emulsifying agent may be also usedin order to improve the rigidity and/or the sealing of the vesicles. Asan example of co-emulsifying agent, mention may be made of cholesteroland derivatives, as for example cholesterol ester charged or neutral ascholesterol sulphate; derivatives with a sterol backbone, for examplederived from plants, such as phytosterol(sitosterol, sigmasterol);ceramides; and mixtures thereof.

Virosomes or their contents may be subject to hydrolysis and physicaldegradation upon storage. According to one embodiment, virosomes may bepreserved for long-term storage by freeze-drying, and reconstituted withan aqueous solution before use. Lyoprotectants such as inulin may beadded prior to lyophilization to help preserve virosome integrity duringlyophilization and upon reconstitution (Wilschut, J. et al., J. LiposomeRes. 17, 2007, 173-182). Preferably, spray freeze-drying is employed(Amorij, J. P. et al. Vaccine 17, 2007, 8707-17).

A virosome-like vesicle of the invention may further comprise atargeting moiety that target said vesicle to a specific cell or tissue.

According to one embodiment, a virosome-like vesicle of the inventionmay further comprise a targeting moiety that target said vesicle to aspecific cell or tissue.

A suitable targeting moiety may be chosen from a cell-surface receptor,a chemokine, a cytokine, a growth-factor, an antibody or an antibodyfragment, a peptide sequence with specificity or specific chargecomplementary to an adhesion molecule such as an integrin. A targetingmoiety may be incorporated into, or attached to the lipid bilayer ofsaid vesicle, by any known techniques of the skilled person in the art.

According to one embodiment, the antigen located to the external surfaceof virosome-like vesicle of the invention may be:

-   -   Covalently linked with a lipid of said virosome-like vesicle, or    -   Intercalated into a lipid bilayer of said virosome-like vesicle        by a peptide transmembrane domain.

According to one embodiment, the antigen may be contained within thevirosome.

Modifications of the antigen of the invention and methods forcross-linking said modified antigen to the external surface of avirosome-like vesicle may be as those described in WO 2004/078099.

According to one embodiment, the antigen may be covalently linked to theexternal surface of a virosome-like vesicle by cross-linking with alipid or a phospholipid. According to another embodiment, the antigenmay be covalently linked to the external surface of a virosome-likevesicle by cross-linking with a carbohydrate. According to anembodiment, a covalently linked antigen may comprise at least oneC-terminally positioned cross-linking residue.

For example, cross-linking residue may be chosen from cysteine (Cys) orlysine (Lys). According to another embodiment a covalently linkedantigen may further comprise at least one spacer residue between saidC-terminally positioned cross-linking residues and a correspondingC-terminal antigen extremity.

A suitable spacer residue may be chosen, for example, from Gly(glycine), Ala (alanine), Ser (serine), Asp (aspartate), Lys (lysine),Gln (glutamine), His (histidine), He (isoleucine) and Leu (leucine)residues. From 2 to 12, in particular from 3 to 10, and moreparticularly from 4 to 8, spacer residues may be linked to form spacersequences. Suitable spacer sequences may be chosen, for example, fromGly-Gly or Lys-Gly.

Cross-linking of the antigen to the surface of a virosome-like vesiclemay be, for example, performed by the use of amphiphilic PEGderivatives, a phosphatidylethanolamine (PE), a phosphatidylcholine(PC), a phosphatidylserine, a cholesterol, or a mixture thereof, readilyincorporated into lipids bilayer. Cross-linking of the antigen to alipid of a virosome-like vesicle of the invention may be performed byany method known to those skilled in the art.

The cross-linking may be operated in a lipid solution and thelipid-peptide conjugate may be subsequently incorporated into avirosome-like vesicle.

According to an embodiment of the invention, the antigen may be linkedto a lipid of a vesicle of the invention, for example, by abifunctionnal succinate linker, in particular a [gamma]-maleinidobutyricacid N-hydroxysuccinimide ester or aN4gamma]-maleimidobutyryloxy-succinimide-ester.

Antigens, lipid linked antigens, phospholipids and adjuvants may beadded to the supernatant formed after solubilization of a virus with adetergent or short-chain phospholipid, and the removal of thenucleocapsid as described above. Virosomes may be then formed, aspreviously described, by detergent removal for example using Bio-BeadsSM-2 (Biorad), Amberlyte XM, or short-chain phospholipid may be removedby dialysis.

Surprinsingly, any conjugate as previously obtained does notsubstantially alter the structure/conformation, nor properties, norfunction(s), of a polypeptide according to the invention, in particularits capacity to trimerize.

Surprisingly, when dissolved in aqueous medium, said conjugates, andthus said polypeptides, remain in dissolved state and stable. Thus,aqueous compositions comprising said conjugates being dissolved in anaqueous medium are expressly encompassed by the present invention.

According to another of its aspects, the instant invention is directedto a pharmaceutical preparation generally comprising any gp41polypeptide according to the invention, whatever its chemical/physicalform, and/or whatever the pharmaceutical adjuvants or excipients.

In a further embodiment, the pharmaceutical preparation comprises atleast as active ingredient a polypeptide according to the invention, ora conjugate as described above, or an expression vector allowing theexpression of the polypeptide of the invention.

Such pharmaceutical preparations possibly comprise an aqueouscomposition according to the invention. A variety of aqueous media maybe used, for pharmaceutical purposes according to the invention, e.g.,water, buffered water, 0.4% saline, 0. 3% glycine, hyaluronic acid andthe like. A pharmaceutical preparation may be sterilized byconventional, well-known sterilization techniques, or may be sterilefiltered.

The pharmaceutical preparations according to the invention may bepackaged for use as are, or lyophilized, the lyophilized preparationbeing combined with a sterile solution prior to administration. Apharmaceutical preparation according to the invention may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, among many others.

A pharmaceutical preparation of the invention may comprise a polypeptideor a trimer thereof or a conjugate thereof and an additionalgp41-derived antigen in an effective amount for treating the patient inneed thereof. Said additional gp41 antigen being distinct from thepolypeptide of the invention. In a more specific embodiment, saidadditional antigen is in the form of a conjugate and even moreparticularly linked to a virosome.

An effective amount is that amount of polypeptide or conjugate accordingto the invention that alone, or together with further doses canstimulate the desired response. An effective amount depends upon avariety of factors, such as the route for administration, whether theadministration is in single or multiple doses, and individual patientparameters including age, physical condition, size, weight, and thestage of the disease. These factors are well known to those of ordinaryskill in the art and can be addressed with no more than routineexperimentation. Therefore, according to an embodiment, a pharmaceuticalpreparation may comprise polypeptides or a trimer thereof or a conjugatethereof of the invention alone or in combination with at least oneadjuvant, as previously described.

Said antigen is distinct/different from the one or those of rgp41according to the invention. Said antigen is also distinct form the oneor those, in particular HA, comprised in said virosomes.

An additional antigen is for instance any part of the gp41 protein, aswell as the gp41 protein, distinct/different from the fragments 540-592and 618-664, the numbering scheme being based upon the prototypicisolate HIV-1 HxB2 clade B in its whole, and analogues thereof.

Said additional antigen may originated from any HIV-1 clade, inpreferred embodiments, said additional antigen originates from clade Bor clade C gp41.

According to an embodiment, a gp41-derived antigen suitable as anadditional antigen for the invention is devoid of fusogenic propertywith respect to cell membrane of target cells.

According to one embodiment, said gp41-derived antigen is covalentlylinked to the external surface of a virosome-like vesicle as previouslydescribed in relation to the conjugates according to the presentinvention comprising virosomes or virosomes-like particles.

According to an embodiment, said gp41-derived antigen is a peptidecalled P1. The peptide P1 corresponds to an amino acid sequence presentin the HIV envelope protein ectodomain gp41 that is located at thesurface of the viral particles before the virus interacts with targetcells. As example, in the HIV-I HxB2 strain, this sequence is comprisedfrom amino acid 649 to amino acid 683, the numbering scheme being basedupon the prototypic isolate HIV-1 HxB2 Clade B strain

In a preferred embodiment, said P1 peptide is described in all or partby a sequence chosen from SEQ ID NO 2, SEQ ID No. 3, SEQ ID No. 6 or ananalogue thereof as described in WO2007/099446, the content of which isincorporated by reference.

As example of a P1 antigen suitable for the invention, it may beenvisioned that the peptide P1 sequence comprising an addition of athree amino acids L-G-C or of a L-S-C spacer at the C-terminal position,as, for example, set forth as SEQ ID NO 4 or SEQ ID No. 5.

In a particular embodiment, the pharmaceutical preparation of thepresent invention is used in immunotherapy, in particular prophylacticimmunotherapy.

A pharmaceutical preparation of the invention comprises a polypeptide ora conjugate of the invention in an effective amount for treating thepatient in need thereof.

In a further embodiment, the pharmaceutical preparation as defined abovecan be used in immunotherapy, in particular prohylactic immunotherapy.

According to another embodiment, a pharmaceutical preparation accordingto the invention may comprise an additional antigen distinct from saidpolypeptide or said conjugate according to the invention as a combinedpreparation for simultaneous, separate or sequential use inimmunotherapy.

According to another of its aspects, the instant invention is alsorelated to a use of at least one gp41 polypeptide, a conjugate or anexpression vector in accordance with the instant invention for themanufacture of a medicament intended to induce an adaptative immuneresponse and/or an innate immune response directed against a gp41protein of a human immunodeficiency virus (HIV).

In a preferred embodiment, the gp41 used polypeptide is represented bySEQ ID No. 19 or SEQ ID No.20 are linked to a virosome.

In a further embodiment the invention is drawn to the use of onepolypeptide, a trimer, an expression vector or a conjugate accordingaccording to the invention and of an additional antigen additionalantigen in the form of a conjugate said conjugate being more preferablya virosome for the manufacture of a medicament intended to induce anadaptative immune response and/or an innate immune response directedagainst a gp41 protein of a human immunodeficency virus, said additionalantigen additional antigen being more preferably described by SEQ ID No2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5 or SEQ ID No.6

Such pharmaceutical preparations may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, antioxidants,preservatives, compatible carriers, adjuvants as described below andoptionally other therapeutic agents.

Adjuvants

According to an embodiment, the immunostimulatory effect of polypeptideor of the conjugate of the invention is obtained, possibly increased byassociating those polypeptide or conjugate with at least one adjuvant.

According to an embodiment, the immunostimulatory effect ofvirosome-like vesicles of the invention may be further increased byassociating those virosome-like vesicles with at least one adjuvant.

Said adjuvant may be encapsulated inside and/or incorporated in thelipid bilayer of, and/or freely combined with said vesicle.

According to one embodiment, a virosome-like vesicle may additionallycomprise at least one adjuvant enhancing and/or mediating an immuneresponse chosen from an innate immune response and/or an adaptativeimmune response. Usable adjuvants may enhance the immunological responseby activating antigen presenting cells (APC), macrophages and/orstimulating specific sets of lymphocytes.

An adjuvant that may convene to the instant invention may be any ligandsuitable for the activation of a pathogen recognition receptor (PRR)expressed in and on dentritic cells (DCs), T-cells, B-cells or otherantigen presenting cells.

Ligands activating the nucleotide-binding oligomerization domain (NOD)receptor pathway may be suited for the purpose of the invention.Adjuvants suitable for these ligands may be muramyl dipeptidederivatives. Ligands activating the Toll-like receptors (TLRs) may alsoconvene for the purpose of the invention. Those receptors are member ofthe PRR family and are widely expressed on a variety of innate immunecells, including DCs, macrophages, mast cells and neutrophils.

As example of ligands activating TLR, mention may be made, for TLR4 ofmonophosphoryl lipid A, 3-O-deacytylated monophosphoryl lipid A, LPSfrom E. coli, taxol, RSV fusion protein, and host heat shock proteins 60and 70, for TLR2 of lipopeptides such asN-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)₃-lysine,peptidoglycan of Staphylococcus aureus, lipoproteins from M.tuberculosis, Sacharomyces cerevisiae zymosan, and highly purified P.gingivalis LPS, for TLR3 of dsRNA, for TLR5 of flagellin, for TLR7synthetic compounds such as imidazoquinolines or for TLR9 of certaintypes of CpG-rich DNA. Other useful adjuvants for the invention may be Thelper epitopes.

A T helper epitope is a peptide usually derived from exogenous proteinsthat have undergone proteolytic degradation and processing within theendocytic pathway of antigen presenting cells (APCs). In those cells theMajor Histocompatibility Complex of class II (MHC II) associates withthose peptides in endosomes. This complex transported to the surface ofthe APCs may interact with a specific T cell receptor of T lymphocytesCD4 leading to their activation. According to the helper epitope, the Tcell response may be of Th1 and/or Th2 type, as known in the art.

As an example of a Th-oriented response epitope one may mention pan DRhelper T cell epitope (PADRE). This epitope is engineered to bind mostcommon HLA-DR molecules with high affinity and to act as a powerfulimmunogen. The PADRE HTL epitope has been shown to augment the potencyof vaccines designed to stimulate a cellular immune response (AlexanderJ. et al., Immunol Res. 18, 1998, 79-92).

According to an embodiment, an adjuvant that may be used with thevirosome-like vesicles of the present invention may be chosen fromaluminum salts, aluminum phosphate gels, mycobacteria such as BCG, M.Vaccae, or corynebacterium parvum, peptides, keyhole limpet hemocyanin,interleukin-2 (IL-2), IL-12, GM-CSF, ligands from the chemokine family,such as RANTES (Regulated upon Activation Normal T cell Expressed andSecreted), a lipoprotein of Gram bacteria, a yeast cell wall component,a double-stranded RNA, a lipopolysaccharide of Gram<“>bacteria,flagellin, a U-rich single-stranded viral RNA, a CpG containing DNA, aSuppressor 6f Cytokine Signalling small interfering RNA (SOCS siRNA),mellitin derived peptides, a pan DR epitope (PADRE) and mixturesthereof.

Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, antioxidants, preservatives,compatible carriers, adjuvants as previously described and optionallyother therapeutic agents.

In one embodiment, a polypeptide or a conjugate according to theinvention may be used for the preparation of a pharmaceuticalpreparation to be administered in the form of a vaccine. Anyimmunization protocol standard in the art may be used. As such anantigenic or immunogenic composition according to the inventioncomprises a polypeptide, a conjugate or a trimer according to theinvention.

Pharmaceutical preparations according to the invention comprisingthereof may be administered systematically by injection or topically bya mucosal route, or a combination thereof.

Injection route may be, for example, intraperitoneal, intradermal,subcutaneous intravascular or intramuscular route.

Any mucosal route may be used, such as genito-urinary route as forexample vaginal route, gastro-intestinal route, anorectal route,respiratory route, upper mucosal tissue, mouth-nasal route and mixturesthereof.

In one embodiment, a pharmaceutical preparation of the invention isprovided as oral dosage forms, such as tablets, capsules (each includingtimed release and sustained release formulations), pills, powders,granules, elixirs, tinctures, solutions, suspensions, syrups andemulsions.

All of these forms are well known to those of ordinary skill in thepharmaceutical art.

According to an embodiment, an object of the invention is to induce witha polypeptide according to the invention, or a conjugate thereof a humansystemic IgA and IgG immune response and/or mucosal IgA and IgG immuneresponse against the HIV virus. Mucosal IgA may be a mixed betweensystemic IgA and secretory IgA response. According to anotherembodiment, an object of the invention is to inhibit or reduce HIVtranscytosis, in particular at musosal level such as the genito-urinarytract, the gastro-intestinal tract, the anorectal route, the respiratorytract, upper mucosal tissues, mouth-nasal route, and combinationsthereof.

Within the meaning of the invention, the expression “adaptative immuneresponse” is intended to refer to an immune response relying upon theactivation of the immune system component, implying specificity andmemory with respect to an antigen or a pathogen.

Such a response may be highly specific toward an antigen or a pathogenand is more effective on second and subsequent encounter, with theantigen or pathogen. Such adaptative immune response may rely on theactivation of lymphocytes, such as T-cells or B-cells.

Within the meaning of the invention, the expression “innate immuneresponse” is intended to refer to a response relying upon thenon-specific recognition system and does not alter upon subsequentencounter with the antigen or pathogen.

Such system may rely upon immune cells such as, for example, monocytesmacrophages or natural killer (NK) or natural killer T (NKT) cells.

According to an embodiment, polypeptides or conjugates according to theinvention may be used for the preparation of a medicament intended toinduce an adaptative immune response and/or an innate immune responsedirected against a gp41 protein of a human immunodeficiency virus.

According to another of its aspects, the instant invention is alsodirected to a method of treatment and/or prophylaxis against a HIVinfection comprising at least a step of administration to an individualin need thereof of an effective amount of a gp41 polypeptide of thepresent invention, a trimer or a conjugate thereof, or an aqueouscomposition as previously defined, all in accordance with the presentinvention.

According to one embodiment of the method of treatment and/orprophylaxis of a HIV infection, the polypeptide or conjugate inaccordance with the invention is administered by injection and/ortopically by the mucosal route, or a combination thereof as previouslyindicated.

Said mucosal route is chosen from genito-urinary tract,gastro-intestinal tract, anorectal route, respiratory tract, uppermucosal tissue, mouth-nasal route and combinations thereof.

In a more specific aspect, within the method of the invention, thepolypeptide is administered in combination with an additional antigendistinct from said gp41-derived antigen as described above. As such, thepreferred method comprises at least the step of administering at least afirst gp41-derived antigen as described by SEQ ID No.19 or SEQ ID No.20or an analogue thereof, and a second gp41-derived antigen as describedby SEQ ID No.4 or SEQ ID No.5 or an analogue thereof.

According to another aspect, the instant invention also relates to a kitfor inducing an immune response against a gp41 protein of a humanimmunodeficiency virus comprising at least a first gp41-derived antigenin the form of a conjugate said conjugate being more preferably avirosome virosome like vesicle of the invention and at least a secondgp41 dervied antigen in the form of a conjugate said conjugate beingmore preferably a virosome virosome like vesicle of the invention, saidfirst and second gp41-derived antigens being different from each other.According to a preferred embodiment the first gp41-derived antigen is apeptide described by SEQ ID No.19 or SEQ ID No.20 or an analoguethereof, the second gp41-derived antigen is a peptide described by SEQID No.4 or SEQ ID No.5 or an analogue thereof.

The instant invention will be further understood with the followingexamples which are presented for illustrating purposes and should not beinterpreted as limiting the scope of the instant invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diagram of the structure of the HIV-1 envelope glycoprotein gp41of clade B, HxB2 strain, presenting the well known epitopes, clustersand regions identified. Open circles correspond to invariant aminoacids, light gray circles correspond to highly conserved amino acids,middle gray circles correspond to moderately variable amino acids, darkcircles correspond to invariant amino acids and amino acids that aresignificantly more variable in one clade than another, are figured asheavily outlined circles. Part of the MPER region, containing theepitopes for the broadly neutralizing antibodies 2F5 and 4E10, andpresent in the P1 peptide of the present invention, is shown on thelower left; this part is absent from the gp41 polypeptide of theinvention. Amino acids from cluster 1 are replaced by a linker in thegp41 protein constructs of the invention.

FIG. 2: cross reactivity of wild type gp41 and derivatives with amonoclonal antibody against gp41, MAb 98.6 (left) and an anti-IL2antibody, AF-202 (right).

FIG. 3: Shows IgA in serum samples of macaques immunized withgp41-virosomes and with P1-virosomes by intramuscular injection (monkeysG2.1 through 2.6), compared with serum of macaques immunized withvirosomes by combined intramuscular and intranasal injection (G3.1though 3.6). Group 1 (G1.1-1.6, placebo) is the control group; theseanimals were immunized intramuscularly with plain influenza virosomeslacking HIV antigens. The data are for serums collected at week 24, andthe OD of pre-immune backgrounds (week 0) have been subtracted form theweek 24 ODs (w24-w0). Sample dilution 1:300.

FIG. 4: IgG measured in serum samples from macaques immunized withgp41-virosomes mixed with P1-virosomes by intramuscular injection(monkeys G2.1 through 2.6), compared with serum of macaques immunizedwith virosomes by combined intramuscular and intranasal injection (G3.1though 3.6). Group 1 (monkeys G1.1-1.6) is the control (placebo) group,immunized intramuscularly with influenza virosomes. The data are forserums collected at week 24, and the OD of pre-immune backgrounds (week0) have been subtracted form the week 24 ODs (w24-w0). Sample dilution1:900.

FIG. 5: Viremia during and after intravaginal challenges withSHIVSF162P3 clade B. Arrows indicate the 13 vaginal challenges with20-30 TCID₅₀, starting one month after the last of four vaccinations,the days after initial challenge being denoted by the oblique numbers.The plasma viral load is indicated by the lines for individual animals;the detection limit of 2.5×10³ copies per mL by the straight line. Lineswith symbols represent individual animals. Groups as in FIGS. 3 and 4.

FIG. 6: Inhibition of HIV-1 transcytosis across epithelial cells bycervicovaginal secretions (CVS). Panel A: strain HIV-1 93BR029 (clade B)Panel B: HIV-1 strain 92BR025 (Clade C) Solid bars: CVS used at a 1:6dilution, open bars 1:10. In the absence of specific IgA antibodiesagainst gp41, transcytosis capability of the HIV-1 is >100% efficient,while in the presence of specific IgA anti-gp41 antibodies, transcytosiscan be reduced >40% with CVS diluted 1:6.

FIG. 7: Inhibition of transcytosis across epithelial cells by CVSsecretions, with or without IgA depletion. Solid bars: with depletion,hatched bars without depletion. When CVS contained mucosal IgAantibodies, inhibition of HIV-1 transcytosis could be observed. When IgAwas depleted from the CVS, transcytosis inhibition wass lost.

FIG. 8: Panel A: Viremia after Clade C virus challenge, with strainSHIV1157ipd3N4, of unvaccinated monkeys.

Panel B: Viremia in monkeys vaccinated with virosomes gp41-derivedantigens from clade B (Group 3 of example 6) and challenged with SHIVclade C, with strain SHIV1157ipd3N4. Eighteen months after the fourthvaccination, animals received a boost and five week later they werechallenged intravaginally 10 times with 10-20 TCID₅₀. The plasma viralload is indicated by the lines for individual animals; note thedetection limit is 10³ copies per mL.

EXAMPLES Example 1 Construction of M0 and M1, polypeptide of theInvention (rgp41) Encoded by SEQ ID No. 28 and SEQ ID NO 21 by MolecularBiology

a) First Step: Construction of gp41-dloop (SEQ ID No.27)

The gp41-delta loop was constructed by PCR.

Design of the Primers

The oligonucleotide primer gp41-Nde (SEQ ID No.22.) and theoligonucleotide primer gp41-Bam1 (SEQ ID No.23) were used to amplify theN-helix and introduce the hydrophilic linker). These oligonucleotideprimers were designed to respectively introduce the sites forrestriction enzymes NdeI and BamHI. The oligonucleotide primer gp41-Bam1was also designed to introduce the oligopeptide linker SGGRGGS (SEQ IDNo.16) to replace the deleted portion of loop.

The oligonucleotide primer gp41-Bam2 (SEQ ID No.24) and theoligonucleotide primer gp41-XhoI (SEQ ID No.25) were used to amplify theC-helix of gp41 by PCR. Those oligonucleotide primers were designed tointroduce the BamHI and the Xho1 enzyme restrictions sites,respectively.

Conditions of PCR

The gp41dloop polynucleotide was amplified from the gp41 matrix (SEQ IDNo.26) by PCR using the above-described oligonucleotide primers. Plasmidwas used at 0. 5 μg/μl primers were used at 10 μM each, and dNTP wereused at 10 mM each. The amplification was conducted using the DNApolymerase DyNazyme from Finnzymes. The amplification was initiated witha denaturing step of 5 minutes at 94° C., following by 30 cycles, eachcomprising a one minute step at 94° C. (denaturing step), a one minutestep at 60° C. (hybridization), and a one minute step at 72° C.(elongation), and the amplification was terminated by a last step of 10minutes at 72° C. The purified PCR products were digested by NdeI(Ozyme, R0111S) and XhoI (Ozyme, R0146S) for insertion in pET21b. ThepET21b vector (Novagen) digested by NdeI and XhoI and the PCR productswere extracted and purified. Ligation was made using Quick ligation kit(New England Biolabs) according to the manufacturer prescriptionresulting in pET21b-gp41dloop.

pET21b-gp41dloop products were transformed in DH5-alpha (Invitrogen).

b) Second Step: Construction of M0gp41C-dloop Clade B (SEQ ID No.28)Encoding M0 Polypeptide (rgp41 According to the Invention)

Two PCRs were performed on the matrix GP41dloop (SEQ ID No.27) to obtainthe M0gp41C_CladeB construct using the Phusion polymerase (Finnzymes).The first reaction was carried out with the primers: GP41B-C-D1 (SEQ IDNo. 29) and GP41B-C-R2 (SEQ ID No. 30)

The second reaction was performed with the primers: GP41B-C-D1 (SEQ IDNo. 29) and GP41B-C-R1 (SEQ ID No. 31).

The positive PCR products were digested using NdeI (Ozyme, R0111S) andXhoI (Ozyme, R0146S) restriction enzymes for the insertion of theGp41C_CladeB encoding gene into pET30b (VWR, 69910-3). The pET30b vectorwas digested using NdeI and XhoI restriction enzymes (Ozyme). The DNAfragments corresponding to Gp41C_CladeB gene and pET30b were extractedand purified (using extraction kit from Macherey-Nagel, 740 609 250).Ligation of these two fragments was done using the Quick ligation kit(New Englands Biolabs Inc, M2200S). 1 μl of the ligation mixture wasused to transform E. coli DH5-alpha (Invitrogen, 12297-016).Transformants were isolated on LB Agar plates with 30 μg/mL kanamycin.Isolated colonies were inoculated in 4 mL of LB medium supplemented with30 μg/mL kanamycin. Cultures were performed overnight at 37° C. and 180rpm. DNA extraction from the corresponding pellets was performedaccording to the protocol given in the Nucleospin Plasmid extraction kitfrom Macherey-Nagel, Ref. 740588-250. They were analyzed by restrictionenzyme digestion and the inserts were sequenced using T7prom (SEQ IDNo.32) and T7term (SEQ ID No.33) primers.

The complete nucleotide sequence of M0gp41dloop-C CladeB was determinedand is represented by SEQ ID No. 28

c) Third Step: Construction of M1gp41C-dloop Clade B (SEQ ID No. 21)Encoding M1 Polypeptide (rgp41 According to the Invention)

The M1gp41C-dloop clade B was constructed by PCR. Two PCRs wereperformed on the matrix Gp41dloopC_CladeB (SEQID No. 28) to obtainM1gp41C-dloop clade B. using the Phusion polymerase (Finnzymes).

The first reaction was done with the primers: GP41B-C-D1 (SEQ ID No. 29)and GP41-C3-R1 (SEQ ID No. 34).

Before the second PCR reaction, purification was done using thenucleospin extract kit (Macherey Nagel, 740609250) with the followingprimers: GP41B-C-D1 (SEQ ID No. 29) and GP41-C3-R2 (SEQ ID No.35).

The positive PCR products were digested using NdeI (Ozyme, R0111 S) andXhoI (Ozyme, R0146S) restriction enzymes for the insertion of theM1gp41dloop-C CladeB encoding gene into pET30b (VWR, 69910-3). ThepET30b vector was digested using NdeI and XhoI restriction enzymes(Ozyme). The DNA fragments corresponding to M1gp41dloop-C CladeB geneand pET30b were extracted and purified (using extraction kit fromMacherey-Nagel, 740 609 250). Ligation of these two fragments was doneusing Quick ligation kit (New Englands Biolabs Inc, M2200S). 1 μl of theligation mixture was used to transform E. coli DH5-alpha (Invitrogen,12297-016). Transformants were isolated on LB Agar plates with 30 μg/mLkanamycin. Isolated colonies were inoculated in 4 mL of LB mediumsupplemented with 30 μg/mL kanamycin. Cultures were performed overnightat 37° C. and 180 rpm. DNA extraction from the corresponding pellets wasperformed according to the protocol given in the Nucleospin Plasmidextraction kit from Macherey-Nagel, Ref. 740588-250. They were analyzedby restriction enzyme digestion and the inserts were sequenced usingT7prom (SEQ ID No.32) and T7term (SEQ ID No.33) primers.

The complete nucleotide sequence of M1gp41dloop-C CladeB was determinedand is represented by SEQ ID No.21.

Example 2 Modified Polypeptide reproduction in E. coli a) Transformation

pET30b-M0gp41dloop-C Clade B or pET30b-M1gp41dloop-C Clade B plasmid wastransformed in the expression E. coli strain BLR (DE3). The expressionof M1gp41dloop-C and M0gp41dloop-C Clade B was driven by a T7 promoter.

b) Expression tests Six cultures of E. coli strain BLR (DE3) carryingthe pET30b-M1gp41dloop-C CladeB or pET30b-M0gp41dloop-C Clade B plasmidwere grown at 37° C. in Luria Broth with 30 mg/ml kanamycin until theoptical density at 600 nm reached 0.6 (spectrophotometer Jasco V-530).The modified polypeptide was induced with 1 mM IPTG (isopropylBD-thiogalactoside), and the culture continued for further 2 hours at37° C. Expression of proteins was checked by separation on SDS-4-12%PAGE.

c) Production

1) Culture One liter of culture of BL21 (DE3)/pET30b-M1gp41dloop-CCladeB or (DE3)/pET30b-M0gp41dloop-C CladeB was grown in Luria Broth at37° C. until the optical density at 600 nm reached the value of 6.0. Theexpression of gp41-engineered loop was induced by 1 mM IPTG, and theculture continued for a further 2 hours at 37° C. The culture wascentrifuged (Centrifuge Beckman Coulter Avanti J20XP with rotor JLA8-1000, 4000×g, 30 min, 4° C.) and the pellet was stored at −80° C.

2) Extraction of M0 or M1 rgp41 Modified Polypeptides

The pellet was resuspended with a sonication buffer (40 mL of Tris-HCl50 mM pH8, NaCl 300 mM). Bacteria were disrupted by a 15 min sonicationstep on ice/ethanol (disintegrator UP200S amplitude 80%, coefficient0.5). Then the suspension was centrifuged at 40 000×g during 30 min at4° C. to separate the soluble proteins (supernatant) from the insolubleproteins (pellet) (Centrifuge Beckman Coulter Avanti J20XP with rotorJA20).

d) Purification of M1 rgp41 Modified Polypeptide

1) Affinity Chromatography

A first step of purification of M1 (encoded by M1gp41dloop-C CladeB, SEQID No. 21) was performed by affinity chromatography on Ni Sepharose™ 6Fast Flow media packed into a XK16/20 column (GE Healthcare) with acolumn volume of 1 mL of medium per 1 L of culture in flasks. Thespecific spacer peptide S allowed the use of an affinity chromatographystep as capture step to recover the soluble fraction of M0 or M1. Thiswas relevant because of the low productivity of the protein. The columnwas equilibrated with a buffer Na-Phosphate 50 mM pH7.5, NaCl 300 mM,β-mercaptoethanol 5 mM. Clarified samples of M1 polypeptide inNa-Phosphate 50 mM pH7.5, NaCl 300 mM, β-mercaptoethanol 5 mM, MgCl₂ 2mM, Leupeptine and Pepstatine 0.1 mM was loaded onto the column at 2ml/min. The column was then washed with equilibration buffer and elutionsteps at 25 mM, 50 mM and 150 mM Imidazole. M1 gp41 was eluted with abuffer Na-Phosphate 50 mM pH7.5, NaCl 300 mM, β-mercaptoethanol 5 mM,Imidazole 300 mM at 4 mL/min. Tween®20 at a final concentration of 0.05%was added on pooled fraction affinity chromatography and a dialysis wasperformed overnight at 4° C. under slow magnetic agitation againstbuffer Na-Phosphate 50 mM pH7.5, NaCl 300 mM, TCEP 1 mM, Tween20 0.05%.Samples were then concentrated 3 times by centrifugation on an Amicon 10kDa concentration unit (Millipore).

2) Size Exclusion ChromatographyA second purification step of M1 wasperformed by Size Exclusion Chromatography using Superdex™ 200 Prepgrade media packed into a XK26/60 column (GE Healthcare) with a columnvolume of 320 ml. The column was equilibrated with a buffer Na-Phosphate50 mM pH7.5, NaCl 300 mM, TCEP 1 mM, Tween20 0.05%. M1 in Na-Phosphate50 mM pH7.5, NaCl 300 mM, TCEP 1 mM, Tween20 0.05% was loaded onto thecolumn at 1.5 ml/min and eluted with the equilibration buffer at 2.5mL/min. According to the calibration curve the protein was eluted assoluble trimers. Therefore, the presence of the spacer comprising thecysteine residue at the C-terminal part of M1 or M0 does not modify theconformational state of the protein.

Example 3 Cross Reactivity

Four proteins were spotted on a nitrocellulose membrane: wt gp41-HA (thenative gp41 fused to an HA tag (GenBank AF348176) in Tris 50 mM pH 8,NaCl 200 mM, Triton X-100 0.1%, 0.1 mg/ml, Gp41-delta loop (SEQ ID No.38) which differs from the native gp41 fragment of FIG. 1 mostly by thereplacement of 25 residues in cluster I with the linker of SEQ ID No. 16in Tris 50 mM pH 8, NaCl 200 mM, glycerol 5%, 0.2 mg/ml, Gp41-engineeredloop (SEQ ID No.37), which differs from the native gp41 fragment of FIG.1 mostly by replacement of 12 residues in cluster I with the linker ofSEQ ID No 16) in Tris 50 mM pH 8, NaCl 200 mM, Imidazol 200 mM, glycerol5%, 0.2 mg/ml, human recombinant IL-2 in Tris 50 mM pH 8, NaCl 200 mM,0.1 mg/ml and a negative control (bovine serum albumin), (FIG. 2).

The membrane was incubated at 37° C., and then put in 20 ml of PBS Tween0.3%, 5% milk for one additional hour under agitation. The 98.6 D (ananti-GP41 human monoclonal antibody from NIBSC, UK) and AF-202 (ananti-human-IL-2 antibody from R & D systems) antibodies were added at afinal concentration of 0.05 μg/ml or 0.5 μg/ml respectively, in 20 ml ofPBS/Tween 20 0.3% -5% milk for one hour with agitation. An appropriateconcentration of anti-IgG peroxidase coupled antibody was added in 20 mlof PBS Tween 0.3%, milk 5% with agitation for one hour, and the blot waswashed three times for 15 minutes each in PBS Tween 0.3%. The twoperoxidase activity was then revealed with with a commercial enhancedchemiluminescence kit (Amersham) and a Kodak film was exposed to theblot, as known in the art.

As shown in FIG. 2, the native Gp41 is strongly recognized by theanti-human IL-2 antibody, the replacement of 25 residues in cluster Ihas abolished the recognition of this protein by the human anti-IL-2antibody, the replacement of 12 residues in cluster I partiallyabrogated the reactivity by the human anti-IL-2 antibody, therecombinant human IL-2 is as expected strongly recognized by the AF-202anti-IL-2 antibody. However, replacement of the 12 or 25 amino acids bya linker does not affect recognition by the anti-gp41 monoclonalantibody 98.6.

Example 4 Solubility Test of the rgp41 Polypeptide

Cultures of E. coli expressing the gp41 polypeptide according to theinvention were centrifuged at 4 000 g at 4° C. during 15 min. Pelletswere suspended in a volume of lysis buffer: phosphate 50 mM pH 7.5, NaCl300 mM, MgCl2 2 mM, beta-mercaptoethanol 5 mM, benzonase 1 microM,pepstatine 1 microM, leupeptine 1 microM to reach OD 600 nm=10. Thesolution was incubated at 4° C. for 30 minutes. Cell lysis was performedby three cycles of freezing/thawing. Soluble and insoluble proteins wereseparated by a 30 min centrifugation at 21 000 g at 4° C. Tenmicroliters of proteins were analyzed to determine the expression andsolubility level by SDS-PAGE 4-12% electrophoresis followed by Coomassieblue staining. The rgp41 polypeptide was found to be present in thesupernatant. Therefore, the protein is soluble.

Example 5 Preparation of Virosome-Like Vesicles Presenting a gp41Polypeptide Produced in E. Coli on the External Surface(rgp41-Virosomes), and Preparation of Virosomes Presenting a SyntheticPeptide on the External Surface (P1-Virosomes)

Virosome-like vesicles were prepared essentially as as described in WO2007/099387. Briefly, influenza A/Singapore/6/86 virus was grown inembryonated eggs and inactivated with beta-propiolacton as known in theart. The virus was dissolved in 100 mM ofoctaethyleneglycolmonodecylether (OEG) in phosphate buffered saline(PBS), and the viral nucleocapsid removed by ultracentrifugation. Thesolubilized membranes, containing 4 mg of hemagglutinin, were mixed with32 mg egg phosphatidylcholine (PC) and 8 mg of phosphatidylethanolamine(PE) dissolved in 2 ml of PBS containing 100 mM OEG. The phospholipidsand the hemagglutinin containing solution as described above were mixedand sonicated for 1 min. This solution was centrifuged for 1 hour at 100000 g and the viral membrane preparation/lipid mixture was sterilized byfiltration.

A polypeptide of the present invention, comprising a spacer and acysteine residue at the C-terminal position (SEQ ID NO 19) wasconjugated through a maleimido-succinimide linker at the N-terminus to aregioisomer of phosphatidylethanolamine (PE) as follows.

Phosphatidylethanolamine (PE) was dissolved in methanol and 0.1% (v/v)triethylamine was added. The solution was then mixed with theheterobifunctional cross-linkerN-[gamma]-maleimidobutyryloxy-succinimide-ester (GMBS), (Pierce ChemicalCompany, Rockford, Ill.) (ratio PE: GMBS=5:1) which was previouslydissolved in dimethylsulfoxide (DMSO) (20 μl). After incubation for 30minutes at room temperature, the solvents were evaporated for 1 h undervacuum in a speedvac centrifuge. The GMBS-PE was then dissolved in 1 mlof PBS containing 100 mM octaethyleneglycol (OEG) (Fluka Chemicals,Switzerland), (PBS-OEG) and the polypeptide of the present inventioncomprising segment S with a cysteine residue at the C-terminal position(SEQ ID NO 19), was added (ratio PE-GMBS:polypeptide=5:1). At this step,the maleimide of the phosphatidylethanolamine-GMBS reacts with thesulfhydryl of the free C-terminal cystein of the gp41 polypeptide. Afterin incubation time of 30 minutes, excess free cystein was added, inorder to quench any remaining free GMBS (ratio Cystein:GMBS=10:1).

The lipid-conjugated polypeptide was added to thehemaggglutinin-containing viral membrane preparation/lipid mixture asdescribed above at a ratio of 1 mg of rgp41 per mg of hemagglutinin, andrgp41-virosomes were formed by detergent removal on SM-2 BioBeads(BioRad, Glattbrugg, Switzerland).

Likewise, the lipid-linked synthetic peptide P1 which aminoacid sequencecorresponds to SEQ ID No. 5 (SQTQQGKNEQELLELDKWASLWNWFDITNWLWYIKLSC(carboxymethyl(1,3-dipalmityol-glycero-2-phophatidylethanolamino))-O Hwas synthesized as the TFA saltSQTQQGKNEQELLELDKWASLWNWFDITNWLWYIKLS-hydroxylcysteine by solid-phaseFmoc chemistry as known in the art, and linked tophosphatidylethanolamine via its C-terminal hydroxylcysteine usingbromoacetyl-phosphoshatidylethanolamine. After purification bypreparative HPLC, and ion exchange to produce its acetate salt, thepeptide was lyophilized, dissolved in PBS-OEG was mixed with the viralmembrane preparation/lipid mixture, at a ratio of 5 mg P1 per mg ofviral hemagglutinin, and P1-virosomes were formed by detergent removal.

Example 6 Immunization of Macaques with a Vaccine Composition Comprisingrgp41-Virosome Like Vesicles of the Invention and P1-Virosomes of theInvention

Immunization of macaques with a vaccine composition comprisingvirosome-like vesicles with rgp41 polypeptide as described above as wellas virosome-like vesicles with gp41 derived antigen peptide P1 locatedat the external surface was carried out as follows.

Three groups of 5 female macaques with an average age of about 5 yearswere used. Four weeks before the first administration of vaccine, allmacaques received intramuscular injections of beta-propiolactoninactivated influenza A Singapore 6/86 (100 μl, 0,01 mg/ml). Thereafter,macaque vaccinations with virosome-like vesicles in aqueous solution (40μg of P1-virosome and 40 μg of rgp41-virosome, 100 μl) were carried outin week 0, 7, 15 and 24. Group 1 (monkeys G1.1 to G 1.6) receivedinfluenza virosomes without gp41 antigens (placebo). Group 2 (G2.1-2.6)received four intramuscular vaccinations with both P1-virosomes andgp41-virosomes at every vaccination, and group 3 (G3.1-3.6) twointramuscular vaccinations (week 0 and 7) followed by two intranasalvaccinations (week 15 and 24), administered as a spray, each time withboth P1-virosomes and gp41-virosomes. One animal in group 3 (3.2) diedfor reasons unrelated to vaccination.

Serum samples were taken at each vaccination time point.

The level of total IgG and IgA antibodies in serum was determinedaccording to the following ELISA protocol. Peptide P1 (SEQ ID NO 5) 100ng/100 μl/wells, or rgp41 of the invention (SEQ ID No. 19, 100 ng/100μl/well) in a bicarbonate buffer 50 mM, pH 9.6 was used to coat ELISAplates (Nunc) overnight at 4° C. Plates were saturated with BSA 2% PBSTween 0.1% for 1 hour 37° C., then washed with PBS-Tween 0.1% buffer.Serums diluted 1/300 for IgA or 1/200 for IgG with PBS Tween 0.1% wereincubated overnight at 4° C. Plates were thereafter rinsed withPBS-Tween 0.1% buffer. For detection of macaque IgG, an anti-macaque IgGgoat antibody couple to biotin (Rockland) (1/15 000) was used followedwith an incubation with streptavidine-HRP (Immunotech) diluted 1/50 000.

For the detection of macaque IgA, an anti-macaque IgA goat antibodycoupled to biotin (Rockland) 1/15 000) was used followed with anincubation with streptavidine-HRP (immunotech) diluted 1/50 000.

A 2F5-IgA monoclonal antibody was used as positive control, followedwith an incubation with an anti-human IgA biotin-labelled goat Fab′2,(0.14 μg/ml final) (Caltag H 14015) and revealed with streptavidine-HRP(1/50,000). A 2F5-IgG monoclonal antibody was used as positive control,followed with a biotinylated anti-human IgG goat Fab′2 (0.1 μg/ml final)(Rockland 609106123) and revealed with streptavidine-HRP (1/50,000).Theantibodies were incubated for 1 hour at 37° C. Colorimetric reaction wastriggered by addition of the substrate TMB, and stopped by addition ofH₂PO₄ 1 M. The optical density (OD) was read at 450 nm.

The results are illustrated in FIG. 3 (gp41-specific IgA in serum) andFIG. 4 (gp41-specific IgG in serum). Results show that female macaquesvaccinated intramuscularly have high levels of specific IgG and IgAanti-gp41 antibodies into their serum. In conclusion, the presence ofIgG as well as IgA antibodies was observed in serum from immunizedfemale macaques. The results revealed that an immune response with IgAmay be obtained with a vaccine of the invention.

To investigate whether vaccination had induced mucosal immunity,cervico-vaginal samples were obtained from all the vaccinated animals ofexample 6 at week 24, by introducing 3 ml of PBS containing antibioticsand protease inhibitors. The samples were centrifuged to remove debris,aliquoted, immediately snap-frozen and stored at −80° C. Mucosal P1antibodies were determined by the ELISA as described above, while cladeB anti-gp41 antibodies were determined according to Tudor et al., 2009,Mucosal Immunol. 2, 412-426. The results were expressed as the number ofanimals having antibody concentrations two times the standard deviation,and compared to the results of serum antibody determinations, expressedin a similar fashion (table I). The serum from monkey 3.2 was excludedfrom analysis.

TABLE I Anti- Serum CVS Antigen body Group 1 Group 2 Group 3 Group 1Group 2 Group 3 P1 IgA 0/6 2/6 0/5 0/6 3/6 4/5 P1 IgG 0/6 6/6 0/5 gp41IgA 0/6 6/6 4/5 0/6 2/6 2/5 gp41 IgG 0/6 6/6 3/5 0/6 2/6 3/5

Additionally, it was observed that the IgA and IgG antibodies were alsoinduced in the genital tract, while IgA was detected in the intestinalcompartiments, even after vaccination by intramuscular injection in theabsence of mucosal adjuvant.

Example 7 Protection Against Heterologous Challenge of the VaccinatedMacaques

The monkeys of example 6 were challenged with live virus as follows:Four weeks after the last vaccination, animals were challengedintra-vaginally 13 times, every 4 to 7 days, with low doses (20-30TCID₅₀) of SHIVSF_(162P3), as shown in FIG. 5. This chimericsimian/clade B human immunodeficiency virus has the pathogenic SIVmac239as a backbone, containing the env (gp120+gp41), tat, rev and vpu genesfrom HIV-1SF162P3. This virus recognizes the receptor CCR5, in contrastto the X4 tropic HxB2 strain used to derive the gp41-construct of theinvention and the peptide P1 of the invention. Therefore, the challengeis with a heterologous virus. The virus was provided by the NIAID(National Institute of Allergy and Infectious diseases), NIH (NationalInstitutes of Health) Bethesda, USA) in 2 mL of PBS.

As shown in FIG. 5, all unvaccinated monkeys (placebo, group 1) wererapidly infected with the virus, with plasma viral loads spiking withintwo weeks at around 10⁶ to 10⁷ copies per ml, as expected (Hessell, A.J. et al. Nat. Med. 15, 951-959). 50% of the monkeys in group 2(intramuscular vaccination) were protected. All animals in group 3(intramuscular/intranasal) were protected; one animal (no. 3.3) had adelayed and low viremia at 800 copies/ml for about one week, and wasnegative thereafter; to confirm, the assay on the samples was repeatedwith a lower detection threshold (FIG. 5). One animal in group 3 diedfor reasons not related to the challenge. These data indicate thatvaccination protects animals against heterologous challenge, althoughgroup 3 has low levels of systemic neutralizing antibodies.

Example 8 Inhibition of Transcytosis and Cross-Clade Protection

To investigate whether vaccination had induced mucosal immunity,cervico-vaginal samples obtained as described in Example 6, wereanalyzed by HIV-1 transcytosis inhibtion assays, performed as previouslydescribed (Bomsel et al., 1997. Nat. Med. 3: 42-47). HIV-I transcytosisacross epithelial cells and the neutralization of transcytosis byantibodies were investigated on the intestinal cell line HT 29 grown asa tight, polarized monolayer for 7 days on a permeable filter support(0.45 μm pore size) forming the interface between two independentchambers, the upper one bathing the apical (luminal) surface of theepithelial monolayer and the lower one bathing the basolateral surface.Prior to transcytosis experiments, epithelial cells were washed, andfurther incubated in RPMI 1640, glutamine, 10% FCS. Cervico-VaginalSecretion (CVS) samples (1/12 and 1/6 dilution) from Group 1 (placebo)or Group 3 (W24; i.m.+i.n.—see example 7 above) were pre-incubated withHIV-1 infected cells (1×10⁶ HIV-1 93BR029 virus (HIV1 clade B) or with92BR025 virus (HIV1 clade C)+PBMCs (Day 7 post infection of activatedPBMCs from healthy individuals with infected with either JRCSF orprimary viruses) for 20 min. at RT. Then, HIV-1 infected cellspre-incubated were added to the apical chamber. Contact between HIV-1infected cells and the epithelial cell monolayer resulted in rapidbudding of the HIV-1-virions, followed by HIV particle internalizationand transcytosis from the apical to the basolateral side of theepithelial cell monolayer. After 2 h, inhibition of transcytosis by CVSwas determined by detection of p24 in the basolateral medium bycommercial ELISA (Coulter, Villepinte, France). During the 2 hrs ofinfected cell contact with epithelial cells, the barrier function of theepithelial monolayer remains intact, precluding penetration of HIV-1infected cells in the monolayer or translocation of HIV infected cellsin the basolateral chamber (1). The HIV-1 transcytosis results are shownin FIG. 6. Clearly, transcytosis of clade B virus was inhibited by theCVS of vaccinated animals. However, surprisingly, vaccination alsoinduced inhibitory activity against clade C virus, as shown by a reducedtranscytosis of HIV-1 respective control (cross-clade protection),suggesting the presence of a shared conformational epitope, as the aminoacid sequence differs between the used viruses

Example 9 Transcytosis is Inhibited by Secretory IgA in the CVS ofVaccinated Animals

Samples of the cervico-vaginal secretions harvested from the animals asdescribed in example 8, were depleted of IgA by incubation withbiotinylated-anti macaque IgA antibodies, as follows. Biotinylatedanti-human IgA (Caltag, france) was bound to streptavidin-agarose(Pierde, France) in a 1:3 weight ratio, and the coupled beads werewashed to remove unbound biotinylated anti-IgA. 30 μl of beads wererotated overnight at 4° C. with CVS (1:6 dilution), and centrifuged for10 min at 1000 g. The resulting supernatant was collected and assayedfollowed by an incubation with streptavidin-agarose beads,(Pierde,France) and a centrifugation to remove the beads. These IgA-depletedsamples were then tested in a transcytosis assay using clade B 93BR029virus, as described in example 8, and compared to samples without IgAdepletion. As shown in FIG. 7, there was little or no inhibitionof HIV-1transcytosis after depletion of IgA, clearly demonstrating the role ofmucosal, rather than serum, IgA in protection against infection.

Example 10 Cross-Clade Protection In Vivo

Since in vitro cross-clade transcytosis inhibition was observed inassays, as indicated above, it was decided to challenge the monkeys fromgroup 3, example 6 (intranasal and intramuscular vaccination with cladeB based virosomes) with a clade C virus. One year after their lastvaccination with the virosomes, the monkeys were still seronegative.They were revaccinated once by intramuscular injection as described inExample 6, and five weeks after vaccination they were challenged 10times, at 4-7 day intervals, with 10-20 TCID₅₀ of SHIV1157ipd3N4 (CladeC, tropism R5, kindly provided by Dr. Ruth Ruprecht, Dana Farber CancerInstitute, USA), At each vaccination time point, blood samples weretaken to determine viremia. Blood samples were taken every 4-7 days for60 days thereafter (FIG. 8). As shown in FIG. 8, the first 40 days afterinfection no vaccinated animals were infected. In a non-vaccinatedcontrol group, 5/6 monkeys were infected at day 11 (FIG. 9), and at day60 all animals were infected. In the vaccinated group, 2/5 monkeysremained uninfected for the 120 days duration of the study, while forthose that were infected, it was significantly delayed respective to thecontrol group.

These surprising data provide clear evidence for cross-clade protectionin vivo.

1. A modified polypeptide comprising three contiguous segments N, L andC represented by the formula N−L−C and comprising: a N-helix region ofgp41 (N), a C-helix region of gp41 (C), and a connecting loop comprisinga synthetic linker (L) between the N and C-helices, the linker replacingamino acids 593-617 of gp41, the numbering scheme being based upon theprototypic isolate HIV-1 HxB2 Clade B strain, said polypeptidecomprising the calveolin-1 neutralizing and 98.6 D epitopes, but not 2F5and 4E10 epitopes, not the fusion peptide, the polypeptide having aminimal immunogenic cross-reactivity with human interleukin 2 (IL2). 2.A polypeptide according to claim 1 further comprising a S fragmentrepresented by the formula N−L−C−S, in which S represents a spacerfragment.
 3. The polypeptide according to claim 1, in which the Nsegment represents the amino acids 540-592 of gp41, the numbering schemebeing based upon the prototypic isolate HIV-1 HxB2.
 4. The polypeptideaccording to claim 1, in which N is described by SEQ ID No. 13 or SEQ IDNo.
 14. 5. The polypeptide according to claim 1, in which the C segmentrepresents the amino acids 618-664 of gp41, the numbering scheme beingbased upon the prototypic isolate HIV-1 HxB2.
 6. The polypeptideaccording to claim 5 in which C is represented by SEQ ID No.15.
 7. Thepolypeptide according to claim 1, in which L is represented by SEQ IDNo.
 16. 8. The polypeptide according to claim 2 in which the S isrepresented by SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.12.
 9. A polypeptide according to claim 1 represented by SEQ ID No. 17or
 18. 10. A polypeptide according to claim 2 described by SEQ ID No. 19or SEQ ID No.
 20. 11. An aqueous composition comprising a polypeptideaccording to claim 1, said polypeptide, forming a trimer in an aqueousmedium.
 12. An aqueous composition according to claim 11, wherein saidtrimer is stable.
 13. A conjugate comprising a polypeptide according toclaim 1, conjugated with a virosome.
 14. A polynucleotide encoding apolypeptide according to claim
 1. 15. A polynucleotide according toclaim 14 described by SEQ ID No. 21 or SEQ ID No.
 28. 16. A polypeptideencoded by a polynucleotide according to claim
 14. 17. A trimercomprising three polypeptides as defined in claim
 1. 18. An expressionvector comprising at least a transcription promoter, a polynucleotideaccording to claim 14 and a transcription terminator.
 19. A host cellcomprising an expression vector according to claim
 18. 20. An antigenicor immunogenic composition comprising: a polypeptide according to claim1; a conjugate comprising the polyaeptide conjugated with a virosome; ora trimer comprising three of the polypeptides.
 21. A pharmaceuticalpreparation comprising: a polypeptide according to claim 1; a conjugatecomprising the polypeptide conjugated with a virosome; a trimercomprising three of the polypeptides; or an expression vector comprisingat least a transcription promoter, a polynucleotide encoding thepolypeptide, and a transcription terminator.
 22. A pharmaceuticalpreparation according to claim 21 comprising an additional antigendistinct from the polypeptide.
 23. A pharmaceutical preparationaccording to claim 22 in which said additional antigen is in the form ofa conjugate.
 24. A pharmaceutical preparation according to claim 23 inwhich said additional antigen is conjugated with a virosome.
 25. Apharmaceutical preparation according to claim 21, in which saidadditional antigen is described by SEQ ID No 2, SEQ ID No 3, SEQ ID No4, SEQ ID No 5 or SEQ ID No.6.
 26. A pharmaceutical preparationaccording to claim 21 for its use in immunotherapy.
 27. A medicamentintended to induce an adaptative immune response and/or an innate immuneresponse directed against a gp41 protein of a human immunodeficencyvirus, the medicament comprising: a polypeptide according to a claim 1;a conjugate comprising the polypeptide conjugated with a virosome; atrimer comprising three of the polypeptides; or an expression vectorcomprising at least a transcription promoter, a polynucleotide encodingthe polypeptide and a transcription terminator.
 28. A conjugate for themanufacture of a medicament intended to induce an adaptative immuneresponse and/or an innate immune response directed against a gp41protein of a human immunodeficency virus, the conjugate comprising: apolypeptide according to claim 1; a conjugate comprising the polypeptideconjugated with a virosome; or a trimer comprising three of thepolypeptides: and an additional antigen described by SEQ ID No. 2, SEQID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No.
 6. 29. A method oftreatment and/or prophylaxis against a HIV infection comprising at leasta step of administration to an individual in need thereof of aneffective amount of polypeptide according to claim 1, a conjugatecomprising the polypeptide conjugated with a virosome or a trimercomprising three of the polypeptides.
 30. The method according to claim29, wherein said effective amount is administered systematically byinjection and/or topically by the mucosal route.
 31. The methodaccording to claims 30, wherein said mucosal route is chosen fromgenito-urinary tract, gastro-intestinal tract, anorectal route,respiratory tract, upper mucosal tissue, mouth-nasal route andcombinations thereof.
 32. The method according to claim 29, wherein saideffective amount is administered in combination with an additionalantigen distinct from said gp41-derived antigen.